1. Field of the Invention
The present invention provides methods for transfecting enteric parasites, transformed enteric parasites and vaccines generated from said transformed enteric parasites.
2. Discussion of the Background
Enteric protozoa cause a variety of diseases in humans. For example, Entamoeba histolytica is the cause of amebiasis, a disease which is surpassed only by malaria and schistosomiasis as a parasitic cause of death (Walsh, J. A. (1986) Rev. Infect. Dis. 8: 228). The parasite's distribution is worldwide, while the preponderance of morbidity and mortality is experienced in Central and South America, Africa and India. Groups at increased risk for severe disease include the very young and old, the malnourished and pregnant women (Armon, P. J., (1978), Brit. J. Ob. Gyn. 85: 264; Walsh, J. A., (1986), Rev. Infect. Dis. 8: 228). For example in Dhaka Bangladesh invasive amebiasis is more common in children of 2-3 years of age and in adults older than 40. The overall malnutrition of the patients may have contributed to the 29% fatality rate despite hospitalization and antiamebic chemotherapy (Wanke, C. et al, (1988), Am. J. Trop. Med. Hyg. 38: 335. E. histolytica is also an important cause of nosocomial (hospital-acquired) infection in developing countries. E. histolytica was found to be the second most common cause of nosocomial diarrhea in a prospective study from the Instituto Nacional de la Nutricion in Mexico City. Mortality in patients with nosocomial diarrhea was 18%, compared to 5% in controls. The preponderance of disease in the developing world is due to fecal-oral spread of infection resulting from complex socioeconomic problems for which there are no immediate solutions. As the improvements in sanitation necessary to prevent the fecal-oral spread of enteric protozoa in the developing world are only slowly being made, control of amebiasis and other diseases is dependent upon advancements in diagnosis, treatment, and immunoprophylaxis.
The pathogenesis of amebiasis begins with cyst formation in the bowel lumen, where unicellular trophozoites undergo nuclear division to form the 4-nucleated cyst. Infection occurs when the cyst is ingested via fecally contaminated food or water. Cysts undergo further nuclear division during excystation leading to the formation of 8 trophozoites. Trophozoites multiply by binary fission. Amebic trophozoites can colonize the bowel lumen, encyst, and/or invade through the intestinal epithelium to cause colitis or liver abscess.
Entamoeba histolytica was named by Schaudinn in 1903 for its ability to destroy human tissues. E. histolytica trophozoites in vitro will kill a wide variety of tissue culture cell lines as well as human neutrophils, T lymphocytes and macrophages. Trophozoite killing of target cells is contact-dependent and extracellular. Killing of host cells by E. histolytica trophozoites in vitro occurs only upon direct contact, which is mediated by an amebic adhesin which recognizes N- and O-linked oligosaccharides (reviewed in McCoy et al, (August 1994) Infect. Immun. 62: in press). This adhesin is specifically inhibited by millimolar concentrations of galactose and N-acetyl-D-galactosamine-(Gal/GalNAc), and has been named the Gal/GalNAc lectin. This lectin is a heterodimer of heavy and light subunits which are encoded by multigene families designated hgl and lgl respectively.
The mechanism of contact-dependent killing by E. histolytica has been the subject of intensive investigation. Intracellular calcium in target cells rises approximately 20-fold within seconds of direct contact by an amebic trophozoite and is associated with membrane blebbing (Ravdin et al, (1988) Infect. Immun. 56: 1505). Cell death occurs 5-15 minutes after the lethal hit is delivered. Extracellular EDTA and treatment of the target cells with the slow sodium-calcium channel blockers verapamil and bepridil (Ravdin et al, (1982) J. Infect. Dis. 154: 27) significantly reduce amebic killing of target cells in suspension. Isolation of amebic pore-forming proteins similar in function to pore-forming proteins of the immune system has been reported by a number of laboratories. (Young et al, (1982) J. Exp. Med. 156: 1677; Lynch et al, (1982) EMBO J 7: 801; Young & Cohn, (1985) J. Cell Biol. 29: 299; Rosenberg et al, (1989) Molec. Biochem. Parasit. 33: 237; Jansson et al, (1994) Science 263: 1440). A purified 5 kDa amoebapore and a synthetic peptide based on the sequence of its third amphiphatic alpha helix have recently been shown to have cytolytic activity for nucleated cells at high concentrations (10-100 .mu.M) (Leippe et al, (1994) Proc. Natl. Acad. Sci. USA 91: 2602). Proteolytic activities of E. histolytica are also believed to be involved in damage of cells and the extracellular matrix of the host. Secreted amebic cysteine proteases cause a cytopathic (as opposed to cytotoxic) effect manifest by cells being released from monolayers in vitro without cell death (Reed et al, (1989) J. Clin. Microbiol. 27: 2772; Tannich et al, (1991) J. Molec. Evol. 34: 272; McKerrow et al, (1993) Ann. Rev. Microbiol. 47: 821).
There are a number of interesting molecules implicated in pathogenesis of enteric protozoan that could be targets for vaccines or therapeutics. The development of DNA transfection methodologies promises to enable genetic validation of their importance in pathogenesis via forward and reverse genetics, enable the production of avirulent enteric protozoa (for use a live vaccines) via genetic "knock-out" of virulence factor genes, as well as set the stage for an understanding of the genetic regulation of the expression of virulence factors during infection and invasion.
To date, little is known about regions required for proper transcription and translation of enteric protozoan genes. While the function of conserved regions identified in the flanking domains of reported genes could be postulated to be involved in regulation of transcription or translation, the lack of a transfection system blocked any attempt to definitively determine the flanking sequences required for gene expression. Thus, the development of a transfection system is required before the genetic elements responsible for proper regulation, promotion, polyadenylation, and ribosomal binding of enteric protozoan genes can be determined.
The development of vaccines against enteric protozoa has been hampered by an incomplete understanding of their pathogenesis. Although several proteins have been identified which appear to be involved in colonization and virulence, in most cases their specific functions and roles in pathogenesis are poorly defined. Enteric protozoa presents a challenge to genetic analysis because there is no known sexual cycle or method to introduce foreign DNA. The ability to manipulate the parasite genome via DNA transfection would allow a more detailed analysis of the factors responsible for virulence as well as enable the production of "attenuated" or avirulent parasites for use as vaccines.